Streptococcal C5a peptidase vaccine

ABSTRACT

Novel vaccines for use against β-hemolytic  Streptococcus  colonization or infection are disclosed. The vaccines contain an immunogenic amount of a variant of streptococcal C5a peptidase (SCP). Also disclosed is a method of protecting a susceptible mammal against β-hemolytic  Streptococcus  colonization or infection by administering such a vaccine. Enzymatically inactive SCP, and polynucleotides encoding these SCP proteins are further disclosed.

This application is a continuation of International Patent ApplicationNo. PCT/US99/28826, filed Dec. 3, 1999 and published in English on Jun.15, 2000 as WO 00/34487, which is a continuation of U.S. applicationSer. No. 09/206,898, filed Dec. 7, 1998 (issued as U.S. Pat. No.6,355,255), which is a continuation-in-part of U.S. Application Ser. No.08/589,756 filed Jan. 22, 1996 (issued as U.S. Pat. No. 5,846,547). U.S.Ser. No. 08/589,756 is incorporated by reference herein.

BACKGROUND OF THE INVENTION

There are several different β-hemolytic streptococcal species that havebeen identified. Streptococcus pyogenes, also called group Astreptococci, is a common bacterial pathogen of humans. Primarily adisease of children, it causes a variety of infections includingpharyngitis, impetigo and sepsis in humans. Subsequent to infection,autoimmune complications such as rheumatic fever and acuteglomerulonephritis can occur in humans. This pathogen also causes severeacute diseases such as scarlet fever, necrotizing fasciitis and toxicshock.

Sore throat caused by group A streptococci, commonly called “strepthroat,” accounts for at least 16% of all office calls in a generalmedical practice, depending on the season. Hope-Simpson, E.,“Streptococcus pyogenes in the throat: A study in a small population,1962-1975,” J. Hyg. Camb., 87:109-129 (1981). This species is also thecause of the recent resurgence in North America and four othercontinents of toxic shock associated with necrotizing fasciitis.Stevens, D. L., “Invasive group A streptococcus infections,” Clin.Infect. Dis., 14:2-13 (1992). Also implicated in causing strep throatand occasionally in causing toxic shock are groups C and G streptococci.Hope-Simpson, E., “Streptococcus pyogenes in the throat: A study in asmall population, 1962-1975,” J. Hyg. Camb., 87:109-129 (1981).

Group B streptococci, also known as Streptococcus agalactiae, areresponsible for neonatal sepsis and meningitis. T. R. Martin et al.,“The effect of type-specific polysaccharide capsule on the clearance ofgroup B streptococci from the lung of infant and adult rats”, J. InfectDis., 165:306-314 (1992). Although frequently a member of vaginalmucosal flora of adult females, from 0.1 to 0.5/1000 newborns developserious disease following infection during delivery. In spite of thehigh mortality from group B streptococcal infections, mechanisms of thepathogenicity are poorly understood. Martin, T. R., et al., “The effectof type-specific polysaccharide capsule on the clearance of Group Bstreptococci from the lung of infant and adult rats,” J. Infect. Dis.,165:306-314 (1992).

Streptococcal infections are currently treated by antibiotic therapy.However, 25-30% of those treated have recurrent disease and/or shed theorganism in mucosal secretions. At present no means is available toprevent streptococcal infections. Historically, streptococcal vaccinedevelopment has focused on the bacterium's cell surface M protein.Bessen, D., et al., “Influence of intranasal immunization with syntheticpeptides corresponding to conserved epitopes of M protein on mucosalcolonization by group A streptococci,” Infect. Immun., 56:2666-2672(1988); Bronze, M. S., et al., “Protective immunity evoked by locallyadministered group A streptococcal vaccines in mice,” Journal ofImmunology, 141:2767-2770 (1988).

Two major problems will limit the use, marketing, and possibly FDAapproval, of an M protein vaccine. First, more than 80 different Mserotypes of S. pyogenes exist and new serotypes continually arise.Fischetti, V. A., “Streptococcal M protein: molecular design andbiological behavior, Clin. Microbiol. Rev., 2:285-314 (1989). Thus,inoculation with one serotype-specific M protein will not likely beeffective in protecting against other M serotypes. The second problemrelates to the safety of an M protein vaccine. Several regions of the Mprotein contain antigenic epitopes which are immunologicallycross-reactive with human tissue, particularly heart tissue. TheN-termini of M proteins are highly variable in sequence and antigenicspecificity. Inclusion of more than 80 different peptides, representingthis variable sequence, in a vaccine would be required to achieve broadprotection against group A streptococcal infection. New variant Mproteins would still continue to arise, requiring ongoing surveillanceof streptococcal disease and changes in the vaccine composition. Incontrast, the carboxyl-termini of M proteins are conserved in sequence.This region of the M protein, however, contains an amino acid sequencewhich is immunologically cross-reactive with human heart tissue. Thisproperty of M protein is thought to account for heart valve damageassociated with rheumatic fever. P. Fenderson et al.,“Tropomyosinsharies immunologic epitopes with group A streptococcal Mproteins, J. Immunol. 142:2475-2481 (1989). In an early trial, childrenwho were vaccinated with M protein in 1979 had a ten fold higherincidence of rheumatic fever and associated heart valve damage. Massell,B. F., et al., “Rheumatic fever following streptococcal vaccination,JAMA, 207:1115-1119 (1969).

Other proteins under consideration for vaccine development are theerythrogenic toxins, streptococcal pyrogenic exotoxin A andstreptococcal pyrogenic exotoxin B. Lee, P. K., et al., “Quantificationand toxicity of group A streptococcal pyrogenic exotoxins in an animalmodel of toxic shock syndrome-like illness,” J. Clin. Microb.,27:1890-1892 (1989). Immunity to these proteins could prevent the deadlysymptoms of toxic shock, but may not prevent colonization bystreptococci.

Thus, there remains a continuing need for an effective means to preventor ameliorate streptococcal infections. More specifically, a need existsto develop compositions useful in vaccines to prevent or amelioratecolonization of host tissues by streptococci, thereby reducing theincidence of strep throat and impetigo. Elimination of sequelae such asrheumatic fever, acute glomerulonephritis, sepsis, toxic shock andnecrotizing fasciitis would be a direct consequence of reducing theincidence of acute infection and carriage of the organism. A need alsoexists to develop compositions useful in vaccines to prevent orameliorate infections caused by all β-hemolytic streptococcal species,namely groups A, B, C and G.

SUMMARY OF THE INVENTION

The present invention provides a vaccine, and methods of vaccination,effective to immunize a susceptible mammal against β-hemolyticStreptococcus. The susceptible mammal could be a human or a domesticanimal such as a dog, a cow, a pig or a horse. Such immunization couldprevent, ameliorate or reduce the incidence of β-hemolytic Streptococcuscolonization in the mammal. The vaccine contains an immunogenic amountof streptococcal C5a peptidase (SCP), wherein the SCP is a variant ofwild-type SCP in combination with a physiologically-acceptable,non-toxic vehicle.

A “variant” of SCP is a polypeptide or oligopeptide SCP that is notcompletely identical to native SCP. Such a variant SCP can be obtainedby altering the amino acid sequence by insertion, deletion orsubstitution of one or more amino acid. The amino acid sequence of theprotein is modified, for example by substitution, to create apolypeptide having substantially the same or improved qualities ascompared to the native polypeptide. The substitution may be a conservedsubstitution. A “conserved substitution” is a substitution of an aminoacid with another amino acid having a similar side chain. A conservedsubstitution would be a substitution with an amino acid that makes thesmallest change possible in the charge of the amino acid or size of theside chain of the amino acid (alternatively, in the size, charge or kindof chemical group within the side chain) such that the overall peptideretains its spacial conformation but has altered biological activity.For example, common conserved changes might be Asp to Glu, Asn or Gln;His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr orGly. Alanine is commonly used to substitute for other amino acids. The20 essential amino acids can be grouped as follows: alanine, valine,leucine, isoleucine, proline, phenylalanine, tryptophan and methioninehaving nonpolar side chains; glycine, serine, threonine, cystine,tyrosine, asparagine and glutamine having uncharged polar side chains;aspartate and glutamate having acidic side chains; and lysine, arginine,and histidine having basic side chains. L. Stryer, Biochemistry (2d ed.)p. 14-15; Lehninger, Biochemistry, p. 73-75.

The amino acid changes are achieved by changing the codons of thecorresponding nucleic acid sequence. It is known that such polypeptidescan be obtained based on substituting certain amino acids for otheramino acids in the polypeptide structure in order to modify or improveantigenic or immunogenic activity. For example, through substitution ofalternative amino acids, small conformational changes may be conferredupon a polypeptide which result in increased activity or enhanced immuneresponse. Alternatively, amino acid substitutions in certainpolypeptides may be used to provide residues which may then be linked toother molecules to provide peptide-molecule conjugates which retainsufficient antigenic properties of the starting polypeptide to be usefulfor other purposes.

One can use the hydropathic index of amino acids in conferringinteractive biological function on a polypeptide, wherein it is foundthat certain amino acids may be substituted for other amino acids havingsimilar hydropathic indices and still retain a similar biologicalactivity. Alternatively, substitution of like amino acids may be made onthe basis of hydrophilicity, particularly where the biological functiondesired in the polypeptide to be generated in intended for use inimmunological embodiments. The greatest local average hydrophilicity ofa “protein”, as governed by the hydrophilicity of its adjacent aminoacids, correlates with its immunogenicity. U.S. Pat. No. 4,554,101.Accordingly, it is noted that substitutions can be made based on thehydrophilicity assigned to each amino acid.

In using either the hydrophilicity index or hydropathic index, whichassigns values to each amino acid, it is preferred to conductsubstitutions of amino acids where these values are ±2, with ±1 beingparticularly preferred, and those with in ±0.5 being the most preferredsubstitutions.

The variant SCP comprises at least seven amino acid residues, preferablyabout 100 to about 1500 residues, and more preferably about 300 to about1200 residues, and even more preferably about 500 to about 1180residues, wherein the variant SCP has at least 50%, preferably at leastabout 80%, and more preferably at least about 90% but less than 100%,contiguous amino acid sequence homology or identity to the amino acidsequence of a corresponding native SCP.

The amino acid sequence of the variant SCP polypeptide correspondsessentially to the native SCP amino acid sequence. As used herein“correspond essentially to” refers to a polypeptide sequence that willelicit a protective immunological response substantially the same as theresponse generated by native SCP. Such a response may be at least 60% ofthe level generated by native SCP, and may even be at least 80% of thelevel generated by native SCP. An immunological response to acomposition or vaccine is the development in the host of a cellularand/or antibody-mediated immune response to the polypeptide or vaccineof interest. Usually, such a response consists of the subject producingantibodies, B cell, helper T cells, suppressor T cells, and/or cytotoxicT cells directed specifically to an antigen or antigens included in thecomposition or vaccine of interest.

The SCP may be a variant of SCP from group A Streptococcus (SCPA), groupB Streptococcus (SCPB), group C Streptococcus (SCPC) or group GStreptococcus (SCPG).

A variant of the invention may include amino acid residues not presentin the corresponding native SCP or deletions relative to thecorresponding native SCP. A variant may also be a truncated “fragment”as compared to the corresponding native SCP, i.e., only a portion of afull-length protein. For example, the variant SCP may vary from nativeSCP in that it does not contain a cell wall insert. SCP variants alsoinclude peptides having at least one D-amino acid.

The variant SCP of the vaccine may be expressed from an isolated DNAsequence encoding the variant SCP. For example, the variant SCP may varyfrom native SCP in that it does not contain a signal sequence or a cellwall insert. The DNA may encode the specificity crevice or the catalyticdomain. In particular the DNA may encode amino acid residue 130, 193,295 or 512 of the catalytic domain, or amino acid residues 260, 261,262, 415, 416 or 417 of the specificity crevice, or encode modificationsat such residues. In particular, the DNA may encode SCPA49D130A,SCPA49H193A, SCPA49N295A, SCPA49S512A, SCPA1D130A, SCPA1H193A,SCPA1N295A, SCPA1S512A, SCPBD130A, SCPBH193A, SCPBN295A, SCPBS512A orΔSCPA49. For the above listing SCPA49H193A means an SCP from group AStreptococci serotype 49, wherein the His at residue number 193 isreplaced with Ala. The SCP of the vaccine may lack enzymatic C5ase orpeptidase activity. The vaccine may also contain an immunologicaladjuvant. The vaccine can be used to prevent infection by group AStreptococcus, group B Streptococcus, group C Streptococcus or group GStreptococcus. The vaccine may comprise an immunogenic recombinantstreptococcal C5a peptidase conjugated or linked to an immunogenicpeptide or to an immunogenic polysaccharide. “Recombinant” is defined asa peptide or nucleic acid produced by the processes of geneticengineering. The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

The streptococcal C5a peptidase vaccine can be administered bysubcutaneous or intramuscular injection. Alternatively, the vaccine canbe administered by oral ingestion or intranasal inoculation.

The present invention further provides isolated and purified SCPpeptides, wherein the SCP is a variant of wild-type SCP and isolated andpurified polynucleotides encoding a variant SCP. For example, the SCPmay include amino acid residue 130, 193, 295 or 512 of the catalyticdomain, or amino acid residues 260, 261, 262, 415, 416 or 417 of thespecificity crevice. The SCP may be SCPA49D130A, SCPA49H193A,SCPA49N295A, SCPA49S512A, SCPA1D130A, SCPA1H193A, SCPA1N295A,SCPA1S512A, SCPBD130A, SCPBH193A, SCPBN295A, SCPBS512A or ΔSCPA49.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Architecture of C5a peptidase from β-hemolytic streptococci. Dindicates an aspartic acid residue; H indicates histidine; S indicatesserine; L indicates leucine; P indicates proline; T indicates threonine;and N indicates asparagine. R₁, R₂, R₃ and R₄ indicate repeatedsequences. The numbers indicate the amino acid residue position in thepeptidase.

FIG. 2. Alignment of the amino acid sequence of SCP from group Astreptococci serotype 49 (SEQ ID NO:1), group A streptococci serotype 12(SEQ ID NO:2), group B streptococci (SEQ ID NO:3) and group Astreptococci serotype 1 (SEQ ID NO:23). The sequences are identicalexcept for the indicated amino acid positions. The triangle (∇)indicates the predicted cleavage point of the signal peptidase. Aminoacids predicted to be in the enzyme's active site are marked byasterisks. Deletions in the amino acid sequence are indicated by dotsand are boxed. The asterisks (*) indicate the amino acid residues of thecatalytic domain.

FIG. 3. Construction of SCP insertion and deletion mutants. Black boxindicates deleted region.

FIG. 4. Single color FACS analysis. Fluorescence data were analyzed bygating on PMNs. A second gate was set to count high staining cellsdefined by the first gate. Air sacs were inoculated with 1×10⁶ CFU.

FIG. 5. Persistence of Wild-type and SCPA⁻ serotype M49 streptococcifollowing intranasal infection.

FIG. 6. Comparison of the ability of SCPA⁻ mutants of serotype M6 GroupA streptococcus to colonize mice following intranasal infection.Compares BALB/c mice (ten in each experimental group) inoculated with2×10⁷ CFU of M6 streptococci. Throat swabs were cultured each day onblood agar plates containing streptomycin. Mice were considered positiveif plates contained one β-hemolytic colony. Data were analyzedstatistically by the χ² test.

FIG. 7. Construction of ΔSCPA49 vaccine and immunization protocol.

FIG. 8. Rabbit antibody neutralizes SCPA activity associated withdifferent serotypes. Bar 1 is a positive control and contained rhC5awhich was not preincubated before exposure to PMNs. Bar 10 is a controlwhich lacks rhC5a. Whole, intact bacteria, preincubated with normalrabbit serum (bar 2, M1 90-131; bar 4, M6 UAB200; bar 6, M12 CS24; bar8, M49 CS101) or preincubated with rabbit anti-S CPA49 serum (bar 3, M190-131; bar 5, M6 UAB200; bar 7, M12 CS24; bar 9, M49 CS 101), wereincubated with 20 μl of 5 μM rhC5a for 45 minutes. Residual rhC5a wasassayed by its capacity to activate PMNs to adhere to BSA-coatedmicrotiter plate wells. Adherent PMNs were stained with crystal violet.

FIGS. 9A and 9B. Serum IgG and secretory IgA responses after intranasalimmunization of mice with the purified ΔASCPA49 protein. Serum andsaliva levels of SCPA49 specific IgG were determined by indirect ELISA.Sera from each mouse were diluted to 1: 2,560 in PBS; saliva was diluted1:2 in PBS. FIG. 9A shows the sIgA experimental results; FIG. 9B showsthe IgG experimental results.

FIGS. 10A and 10B. Comparison of the ability of serotype M49streptococci to colonize immunized and non-immunized CD1 female mice.Each experimental group contained 13 mice which were infectedintranasally (i.n.) with 2.0×108 CFU. The data were analyzedstatistically by the χ² test. FIGS. 10A and 10B show the results of therepeated experiment. CFU. The data were analyzed statistically by the χ²test. FIGS. 10A and 10B show the results of the repeated experiment.

FIG. 11. Competitive ELISA Comparison of wild-type and variant SCPbinding to polyclonal antibody. Plate antigen is recombinant wild-typeSCPA49 (100 ng/well). Competing antigen is indicated by the legend.

FIG. 12. Competitive ELISA Comparison of SCPA1, SCPA49 and SCPB bindingto polyclonal antibody. Plate antigen is recombinant wild-type SCPA49(100 ng/well). Competing antigen is indicated by the legend. SCPA1 andSCPA49 used in the experiments depicted in this Figure comprised Asn³²through His¹¹³⁹. SCPB used in the experiments depicted in this Figurewas made according to Chmouryguina, I. et al., “Conservation of the C5aPeptidase Gene in Group A and B Streptococci”, Infect. Immun.,64:2387-2390 (1996).

DETAILED DESCRIPTION OF THE INVENTION

An important first line of defense against infection by many bacterialpathogens is the accumulation of phagocytic polymorphonuclear leukocytes(PMNs) and mononuclear cells at the site of infection. Attraction ofthese cells is mediated by chemotactic stimuli, such as host factors orfactors secreted by the invading organism. The C5a chemoattractant ispivotal to the stimulation of this inflammatory response in mammals. C5ais a 74 residue glycopeptide cleaved from the fifth component (C5) ofcomplement. Phagocytic cells respond in a directed manner to a gradientof C5a and accumulate at the site of infection. C5a may be the mostimmediate attractant of phagocytes during inflammation. As PMNsinfiltrate an inflammatory lesion they secrete other chemokines, such asIL8, which further intensify the inflammatory response.

Streptococcal C5a peptidase (SCP) is a proteolytic enzyme located on thesurface of pathogenic streptococci where it destroys C5a, as C5a islocally produced. SCP specifically cleaves the C5a chemotaxin at the PMNbinding site (between His⁶⁷-Lys⁶⁸ residues of C5a) and removes the sevenmost C-terminal residues of C5a. This cleavage of the PMN binding siteeliminates the chemotactic signal. Cleary, P., et al., “StreptococcalC5a peptidase is a highly specific endopeptidase,” Infect. Immun.,60:5219-5223 (1992); Wexler, D. E., et al., “Mechanism of action of thegroup A streptococcal C5a inactivator,” Proc. Natl. Acad. Sci. USA,82:8144-8148 (1985).

SCP from group A streptococci is a subtilisin-like serine protease withan M_(r) of 124,814 da and with a cell wall anchor motif which is commonto many Gram positive bacterial surface proteins. The architecture ofC5a peptidase is given in FIG. 1. The complete nucleotide sequence ofthe streptococcal C5a peptidase gene of Streptococcus pyogenes has beenpublished. Chen, C., and Cleary, P., “Complete nucleotide sequence ofthe streptococcal C5a peptidase gene of Streptococcus pyogenes,” J.Biol. Chem., 265:3161-3167 (1990). In contrast to Subtilisins, SCP has avery narrow substrate specificity. This narrow specificity is surprisingin light of the marked similarities between their catalytic domains.Cleary, P., et al., “Streptococcal C5a peptidase is a highly specificendopeptidase,” Infect. Immun., 60:5219-5223 (1992). Residues involvedin charge transfer are conserved, as are residues on both sides of thebinding pocket. However, the remaining amino acid sequence of SCP isunrelated to that of Subtilisins. More than 40 serotypes of Group Astreptococci were found to produce SCP protein or to harbor the gene.Cleary, P., et al., “A streptococcal inactivator of chemotaxis: a newvirulence factor specific to group A streptococci,” in Recent Advancesin Streptococci and Streptococcal Disease p.179-180 (S. Kotami and Y.Shiokawa ed.; Reedbooks Ltd., Berkshire, England; 1984); Podbielski, A.,et al., “The group A streptococcal virR49 gene controls expression offour structural vir regulon genes,” Infect. Immun., 63:9-20 (1995).

The active site of SCP is composed of the charge transfer system and thespecificity crevice. The charge transfer system, also called thecatalytic domain, contains residues Asp¹³⁰, His¹⁹³ ASN295 and Ser⁵¹²(FIGS. 1 and 2). A modification, i.e., a deletion, insertion orsubstitution, of any one of these amino acids will inactivate theenzyme. The specificity crevice, on the other hand, is predicted to beformed by Ser²⁶⁰, Phe²⁶¹, Gly²⁶², Ile⁴¹⁵, Tyr⁴¹⁶ and Asp⁴¹⁷.Modification by substitution of these amino acids could change thesubstrate specificity of the enzyme or eliminate proteolytic activityaltogether. Modification by deletion of these amino acids would alsoinactivate the enzyme. The catalytic domain depends on the tertiarystructure of the protein that is created when the mature enzyme foldsinto its active state. This domain is not formed from a contiguouslinear array of amino acid residues. Alternatively, modification mayalso reduce binding of variant SCP to the substrate. Binding may bereduced by 50%, 70% or even 80%.

A C5a peptidase enzyme associated with group B streptococci has alsobeen identified. Hill, H. R., et al., “Group B streptococci inhibit thechemotactic activity of the fifth component of complement,” J. Immunol.141:3551-3556 (1988). Restriction mapping and completion of the scpBnucleotide sequence showed that scpB is 97-98% similar to scpA. See FIG.2 for comparison of the amino acid sequence of SCP from group Astreptococci serotype 49, group A streptococci serotype 12, group Bstreptococci and group A streptococci serotype 1 (SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:23, respectively). More than 30 strains,representing all serotypes of group B streptococci carry the scpB gene.Cleary P. P., et al. “Similarity between the Group B and A streptococcalC5a Peptidase genes,” Infect. Immun. 60:4239-4244 (1992); Suvorov A. N.,et al., “C5a peptidase gene from group B streptococci,” in Genetics andMolecular Biology of Streptococci, Lactococci, and Enterococci p.230-232 (G. Dunny, P. Cleary and L. McKay (ed.); American Society forMicrobiology, Washington, D.C.; 1991).

Human isolates of groups G and C streptococci also harbor scpA-likegenes. Some group G strains were shown to express C5a specific proteaseactivity on their surface. Cleary, P. P., et al., “Virulent humanstrains of group G streptococci express a C5a peptidase enzyme similarto that produced by group A streptococci,” Infect. Immun., 59:2305-2310(1991). Therefore, all serotypes (>80) of group A streptococci, group Bstreptococci, group C streptococci and group G streptococci produce theSCP enzyme.

SCP assists streptococci to colonize a potential infection site, such asthe nasopharyngeal mucosa, by inhibiting the influx of phagocytic whitecells to the site of infection. This impedes the initial clearance ofthe streptococci by the host. The impact of SCP on inflammation, C5aleukocyte chemotaxis and streptococcal virulence was examined usingstreptococcal strains with well-defined mutations in the proteasestructural gene. SCP variants were constructed by targeted plasmidinsertion and by replacement of the wild-type gene with scpA containinga specific internal deletion. Variants lacked C5a protease activity anddid not inhibit the chemotactic response of human or mouse PMNs to C5ain vitro.

A mouse connective tissue air sac model was used to confirm that SCPretards the influx of phagocytic cells and clearance of streptococcifrom the site of infection. A connective tissue air sac is generated byinjecting a small amount of air and PBS (with or without streptococci init) with a 25-gauge needle under the skin on the back of a mouse. Boyle,M. D. P. et al., “Measurement of leukocyte chemotaxis in vivo,” Meth.Enzymol., 162:101:115 (1988). At the end of the experiment, the micewere euthanized by cervical dislocation, the air sacs dissected from theanimals, and the air sacs homogenized in buffer. An advantage of the airsac model is that the air sac remains inflated for several days and freeof inflammation, unless an irritant is injected. Thus, injected bacteriaand the resulting inflammatory response remains localized over shortperiods of infection.

The air sac model was modified to compare clearance of wild-type SCP⁺andSCP streptococci (i.e., group A streptococci which carried a variantnon-functional form of SCP), and to analyze the cellular infiltrate atan early stage of infection. Tissue suspensions were assayed for viablestreptococci on blood agar plates and the cellular infiltrate wasanalyzed by fluorescent cell sorting (FACS). In FACS analysis,individual cells in suspension are labelled with specific fluorescentmonoantibodies. Aliquots of labelled cells are injected into a FACScan™flowcytometer, or fluorescent cell sorter, which counts cells based ontheir unique fluorescence. The experiments using the air sac modelindicated that streptococci that were SCP⁺were more virulent thanstreptococci that were SCP.

A study was performed to measure production of human antibody, both IgGand IgA, against SCP in human sera and saliva. O'Connor, SP, et al.,“The Human Antibody Response to Streptococcal C5a Peptidase,” J. Infect.Dis. 163:109-16 (1991). Generally, sera and saliva from young,uninfected children lacked antibody to SCP. In contrast, most sera andsaliva specimens from healthy adults had measurable levels of anti-SCPIgG and SCP-specific secretory IgA (anti-SCP sIgA). Paired acute andconvalescent sera from patients with streptococcal pharyngitis possessedsignificantly higher levels of anti-SCP IgG than did sera from healthyindividuals. Sera containing high concentrations of anti-SCPimmunoglobulin were capable of neutralizing SCP activity. Detection ofthis antibody in >90% of the saliva specimens obtained from children whohad recently experienced streptococcal pharyngitis demonstrated thatchildren can produce an antibody response.

Even though the human subjects produced IgG and IgA against SCP inresponse to a natural streptococcal infection, it was not known whetherthe anti-SCP immunoglobulin provides any protection against infection.Further, it was not known if the SCP protein could act as a vaccineagainst β-hemolytic streptococcal colonization or infection. First, astudy was performed to examine the role of SCP in colonization of thenasopharynx. Following intranasal infection with live group Astreptococci, throat cultures were taken daily for up to ten days.Wild-type and isogenic SCP-deficient mutant streptococci were comparedfor the ability to persist in the throat over this ten day period. Aspredicted, the SCP-deficient mutant streptococci were cleared from thenasopharynx more rapidly.

The same intranasal mouse model was used to test the capacity of SCP toinduce immunity that will prevent colonization. A variant form of therecombinant scpA49 gene beginning at the nucleotide that encodes Thr⁶³was cloned. This variant is referred to as ΔSCPA49, and is 2908 bp inlength (see Example 4 below). Variant SCP protein was purified from anE. coli recombinant by affinity chromatography. Sera from rabbitsvaccinated intradermally with this protein preparation neutralized SCPactivity in vitro. Purified protein (40 μg) was administeredintranasally to mice over a period of five weeks. Immunized mice clearedstreptococci in 1-2 days; whereas, throat cultures of non-immunized miceremained positive for up to 10 days. The experiment was repeated onthree sets of mice, vaccinated with three separate preparations of a SCPprotein.

Further experiments were performed to determine whether immunization ofan animal with a single antigen would prevent colonization by severalserotypes. ΔSCPA49 was cloned into an expression vector and expressed inE. coli. The affinity purified variant ΔSCPA49 protein proved to behighly immunogenic in mice and rabbits. Although the purified variantΔSCPA49 immunogen lacked enzymatic activity, it induced high titers ofrabbit antibodies that were able to neutralize peptidase activityassociated with M1, M6, M12 and M49 streptococci in vitro. Thisconfirmed that anti-peptidase antibodies lack serotype specificity. Foursets of mice were then intranasally immunized with the purified variantΔSCPA49 and each was challenged with a different serotype of group Astreptococcus. The immunization of mice with ΔSCPA49 protein stimulatedsignificant levels of specific salivary sIgA and serum IgG antibodiesand reduced the potential of wild-type M1, M2, M6, M11 and M49streptococci to colonize. These experiments confirm that immunizationwith streptococcal C5a peptidase vaccine is effective in preventing thecolonization of the nasopharynx.

Experiments were also performed to develop variant SCPs from an M1 OF⁻strain and from the M49 OF⁺ strain. Since active SCP could be harmful tothe host, it was important that the variant proteins lacked enzymaticactivity. Amino acids that are required for catalytic activity werereplaced with those expected to inactivate the enzyme.

Two properties of the variant proteins were evaluated. First, thespecific activities of the wild-type and variant proteins weredetermined by PMN adherence assay. These experiments indicated that thesubstituted amino acids reduced enzymatic activity by greater than 90%.Second, the variant proteins were also compared to the wild-type proteinfor their capacity to bind antibody directed against the wild-typeenzyme. Competitive ELISA assays were used for this purpose. The resultsindicated that the amino acid substitutions did not alter the ability ofantibody to bind to the variant proteins.

All earlier protection studies had been performed by administeringaffinity purified ΔSCPA49 protein intranasally without adjuvant.Intramuscular or subcutaneous (SQ) injection of antigens, however, ishistorically a preferred, more accepted method of vaccine delivery.Therefore, experiments were performed to test whether SQ injections ofΔSCPA with monophosphoryl lipid A (MPL) and alum (AlPO₄) induced aprotective immune response and whether that response reducedcolonization when the challenge strain of group A streptococcus differedin serotype from the source of the SCPA vaccine. The capacity ofimmunized mice to clear streptococci from the oral-nasal pharyngealmucosa was evaluated by throat culture or by sampling dissected nasaltissue.

The number of streptococci associated with nasal tissue decreased withtime, as expected, and the decrease was more rapid and complete in miceimmunized with SCPA antigen. The results confirmed that a single SCPAantigen can induce protection against heterologous serotypes. Protectionis afforded by antibody that neutralizes peptidase activity on thebacterial surface. This increases the influx of phagocytes within a fewhours from the time streptococci are deposited on mucosal tissue. Rapidclearance of streptococci by phagocytes is presumed to preventsubsequent multiplication and persistence of the bacteria. Thus, SQinjection of SCPA antigen with adjuvant consistently induced a vigorousantibody response.

The present invention thus provides a vaccine for use to protect mammalsagainst β-hemolytic Streptococcus colonization or infection. In oneembodiment of this invention, as is customary for vaccines, the variantstreptococcal C5a peptidase can be delivered to a mammal in apharmacologically acceptable vehicle. Vaccines of the present inventioncan also include effective amounts of immunological adjuvants, known toenhance an immune response.

The SCP can be conjugated or linked to another peptide or to apolysaccharide. For example, immunogenic proteins well-known in the art,also known as “carriers,” may be employed. Useful immunogenic proteinsinclude keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA),ovalbumin, human serum albumin, human gamma globulin, chickenimmunoglobulin G and bovine gamma globulin. Useful immunogenicpolysaccharides include group A Streptococcal polysaccharide,C-polysaccharide from group B Streptococci, or the capsularpolysaccharides of Streptococcus pnuemoniae or group B Streptococci.Alternatively, polysaccharides or proteins of other pathogens that areused as vaccines can be conjugated to, linked to, or mixed with SCP.

Further provided are isolated and purified nucleic acid molecules, e.g.,DNA molecules, comprising a preselected nucleic acid segment whichencodes at least a portion of a Streptococcal C5a peptidase, i.e., theyencode SCP or a variant thereof as described herein, e.g., SCPA49S512A,SCPA49D130A, SCPA49N295A, SCPA1S512A, SCPA1D130A, SCPA1N295A, ΔSCPA49,SCPBS512A, SCPBD130A, SCPBH193A or SCPBN295A, or any combination ofthese mutations. For example, the invention provides an expressioncassette comprising a preselected DNA segment which codes for an RNAmolecule which is substantially identical (sense) to all or a portion ofa messenger RNA (“target” mRNA), i.e., an endogenous or “native” SCPmRNA. The preselected DNA segment in the expression cassette is operablylinked to a promoter. As used herein, “substantially identical” insequence means that two nucleic acid sequences have at least about 65%,preferably about 70%, more preferably about 90%, and even morepreferably about 98%, contiguous nucleotide sequence identity to eachother. Preferably, the preselected DNA segment hybridizes underhybridization conditions, preferably under stringent hybridizationconditions, to a nucleic acid molecule encoding the corresponding nativeSCP.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, about 90%, about 95%, and about 99%. Most preferably,the object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) wherein the composition consists essentially of a singlemacromolecular species.

As used herein, the term “recombinant nucleic acid” or “preselectednucleic acid,” e.g., “recombinant DNA sequence or segment” or“preselected DNA sequence or segment” refers to a nucleic acid, e.g., toDNA, that has been derived or isolated from any appropriate source, thatmay be subsequently chemically altered in vitro, so that its sequence isnot naturally occurring, or corresponds to naturally occurring sequencesthat are not positioned as they would be positioned in a genome whichhas not been transformed with exogenous DNA. An example of preselectedDNA “derived” from a source, would be a DNA sequence that is identifiedas a useful fragment within a given organism, and which is thenchemically synthesized in essentially pure form. An example of such DNA“isolated” from a source would be a useful DNA sequence that is excisedor removed from said source by chemical means, e.g., by the use ofrestriction endonucleases, so that it can be further manipulated, e.g.,amplified, for use in the invention, by the methodology of geneticengineering.

Recovery or isolation of a given fragment of DNA from a restrictiondigest can employ separation of the digest on polyacrylamide or agarosegel by electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. See Lawn et al., NucleicAcids Res., 9,6103 (1981), and Goeddel et al., Nucleic Acids Res.,8,4057 (1980). Therefore, “preselected DNA” includes completelysynthetic DNA sequences, semi-synthetic DNA sequences, DNA sequencesisolated from biological sources, and DNA sequences derived from RNA, aswell as mixtures thereof.

As used herein, the term “derived” with respect to a RNA molecule meansthat the RNA molecule has complementary sequence identity to aparticular DNA molecule.

Nucleic acid molecules encoding amino acid sequence variants of a SCPare prepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring amino acid sequence variants) or preparationby oligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the SCP.

To immunize a subject, the variant SCP, is administered parenterally,usually by intramuscular or subcutaneous injection in an appropriatevehicle. Other modes of administration, however, such as oral deliveryor intranasal delivery, are also acceptable. Vaccine formulations willcontain an effective amount of the active ingredient in a vehicle. Theeffective amount is sufficient to prevent, ameliorate or reduce theincidence of β-hemolytic Streptococcus colonization in the targetmammal. The effective amount is readily determined by one skilled in theart. The active ingredient may typically range from about 1% to about95% (w/w) of the composition, or even higher or lower if appropriate.The quantity to be administered depends upon factors such as the age,weight and physical condition of the animal or the human subjectconsidered for vaccination. The quantity also depends upon the capacityof the animal's immune system to synthesize antibodies, and the degreeof protection desired. Effective dosages can be readily established byone of ordinary skill in the art through routine trials establishingdose response curves. The subject is immunized by administration of theSCP in one or more doses. Multiple doses may be administered as isrequired to maintain a state of immunity to streptococci.

Intranasal formulations may include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspension, solutions, emulsions, syrups or elixirs, or may bepresented dry in tablet form or a product for reconstitution with wateror other suitable vehicle before use. Such liquid preparations maycontain conventional additives such as suspending agents, emulsifyingagents, non-aqueous vehicles (which may include edible oils), orpreservative.

To prepare a vaccine, the purified SCP can be isolated, lyophilized andstabilized. The SCP peptide may then be adjusted to an appropriateconcentration, optionally combined with a suitable vaccine adjuvant, andpackaged for use. Suitable adjuvants include but are not limited tosurfactants, e.g., hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′-N-bis(2-hydroxyethyl-propane di-amine),methoxyhexadecyl-glycerol, and pluronic polyols; polanions, e.g., pyran,dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g.,muramyl dipeptide, MPL, aimethylglycine, tuftsin, oil emulsions, alum,and mixtures thereof. Other potential adjuvants include the B peptidesubunits of E. coli heat labile toxin or of the cholera toxin. McGhee,J. R., et al., “On vaccine development,” Sem. Hematol., 30:3-15 (1993).Finally, the immunogenic product may be incorporated into liposomes foruse in a vaccine formulation, or may be conjugated to proteins such askeyhole limpet hemocyanin (KLH) or human serum albumin (HSA) or otherpolymers.

The application of SCP for vaccination of a mammal against colonizationoffers advantages over other vaccine candidates. Prevention ofcolonization or infection by inoculation with a single protein will notonly reduce the incidence of the very common problems of strep throatand impetigo, but will also eliminate sequelae such as rheumatic fever,acute glomerulonephritis, sepsis, toxic shock and necrotizing fasciitis.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1 Construction and In Vitro Analysis of Insertion and DeletionMutants in scpA49 and scpA6

a) Bacterial strains and culture conditions. S. pyogenes strain CS101 isa serotype M49, and serum opacity positive (OF⁺) strain. CS159 is aclinical isolate with a deletion which extends through the M genecluster and scpA. A spontaneous, streptomycin resistant derivative ofstrain CS 101, named CS101Sm, was selected by plating streptococci froma stationary phase culture on tryptose blood agar containingstreptomycin (200 μg/ml). Streptococcal strains CS210 and CS463 arespontaneous streptomycin resistant derivatives of OF⁺, class II,serotype M2, and M11 strains, respectively. Streptococcal strains 90-131and UAB200 are spontaneous streptomycin resistant derivatives of OF⁻,class I, serotype M1 and M6 human isolates of group A streptococci,respectively.

CS 101::pG⁺host5 is strain CS 101 with pG⁺host5 integrated into thechromosome at an unknown location, but outside scpA and the emm genecluster. Escherichia coli strain ER1821 (from New England Biolabs, Inc.Beverly, Mass.) was used as the recipient for the suicide vector,plasmid pG⁺host5. Plasmid pG⁺host5 was obtained from Appligene, Inc.Pleasanton, Calif. Streptococci were grown in Todd-Hewitt brothsupplemented with 2% neopeptone or 1% yeast extract, or on tryptose agarplates with 5% sheep blood. E. coli strain ER1821 containing plasmidpG⁺host5 was grown in LB broth with erythromycin (300 μg/ml).Streptococci with plasmid pG⁺host5 were cultured in Todd-Hewitt brothwith 1% yeast extract (THY) containing 1 μg/ml of erythromycin (Erm).

SCP refers to streptococcal C5a peptidase from β-hemolytic Streptococcusgenerally. SCPA1, SCPA12, SCPA49, SCPA6 are the specific peptidases fromgroup A Streptococcus M serotype 1, 12, 49 and 6 strains, respectively.The term scpA refers to the gene encoding SCP from group A streptococci.ScpA1, scpA12, scpA6 and scpA49 are the genes encoding the SCPA1,SCPA12, SCPA49 and SCPA6 peptidases. SCPB and scpB refer to thepeptidase and gene from group B streptococci. The amino acid sequencesfor SCPA49 (SEQ ID NO:1), SCPA12 (SEQ ID NO:2), SCPA1 (SEQ ID NO:23 andSCPB (SEQ ID NO:3) are given in FIG. 2.

b) Construction of scpA49 insertion mutant. Well-defined insertionmutants of scpA49 were constructed using plasmid insertion and genereplacement methods. An internal scpA49 BglII-BamHI fragment, theinsertion target, was ligated into the thermosensitive shuttle vectorpG⁺host5 to form plasmid pG::scpA1.2 and transformed into E. coli ER1821(FIG. 3). The pG⁺host5 vector contains an E. coli origin of replicationthat is active at 39° C., a temperature sensitive Gram positive originof replication (active at 30° C. and inactive at 39° C. instreptococci), and an erythromycin resistance gene for selection. Hightemperature forces the plasmid to integrate into the chromosomal DNA ofgroup A streptococci by homologous recombinant at frequencies rangingfrom 10⁻² to 10⁻³.

Recombinant plasmid DNA pG::scpA1.2 was electroporated into CS 101recipient cells. Transformants were selected on THY-agar platescontaining 1 μg/ml erythromycin at 30° C. Chromosomal integrants whichresulted from recombination between the plasmid insert and thechromosomal scpA49 were selected by erythromycin resistance at 39° C.Two insertion mutants, M14 and M16, were analyzed. EmrS revertants ofstrain M14 and M16 were obtained by passage in THY without antibiotic at30° C. and finally plated at 37° C. without Erm selection. Colonies thathad lost the plasmid were isolated to confirm that the mutant phenotyperesulted from insertion of the plasmid into scpA49, rather than from asimultaneous unrelated mutation.

c) Construction of the scpA6 insertion mutants. The scpA6 insertionmutant AK1.4 was constructed as described in section (b) above.Recombinant plasmid DNA, pG::scpA1.2, contains an internal BglII-HindIIIfragment of scpA gene. This plasmid was electroporated into UAB200recipient cells and transformants were selected on THY agar platescontaining erythromycin at 30° C. A chromosomal integrant ofpG::scpA1.2, strain AK1.4, which resulted from recombination between theplasmid insert and the chromosomal scpA6 was selected by growth on agarmedium containing erythromycin at 39° C. Insertion into scpA6 wasconfirmed by Southern blotting using scpA as the probe, and PCR using anM13 universal primer (5′-GTAAAACGACGGCCAGT-3′) (SEQ ID NO:6), specificfor the plasmid, and an scpA For835 primer (5′-AAGGACGACACATTGCGTA-3′)(SEQ ID NO:7), specific for the chromosomal scpA of GAS.

d) Introduction of a defined deletion into scpA (FIG. 3). A mutantstrain with a defined deletion internal to scpA49 was constructed toeliminate the possibility that insertions in scpA49 could be polar andreduce expression of downstream genes, unknown genes which could alsocontribute to the organism's virulence. First, a defined deletion inBglII-HindIII fragment of scpA was produced by inside-out PCR withprimer 1 (5′-GGGGGGGAATTCGTAGCGGGTATCATGGGAC-3′), SEQ ID NO:4, andprimer 2 (5′-GGGGGGGAATTCGGGTGCTGCAATATCTGGC-3′), SEQ ID NO:5.Underlined nucleotides correspond to scpA sequences with coordinates2398 and 2322, respectively, and the bold faced nucleotides correspondto a EcoRI recognition site. The primers were selected to produce anin-frame deletion in the scpA gene. These primers copy plasmid DNA inopposite directions and define the boundaries of the deletion. Innis, M.A., et al., eds., PCR Protocols A Guide to Methods and Applications(Academic Press, 1990). Plasmid pG::scpA1.2 DNA was used as template.

The amplified product was digested with EcoRI and ligated to plasmidpG⁺host5. The resulting plasmid pG::ΔscpA1.1 contained an 76 bp deletioninternal to scpA. This in-frame deletion removed 25 amino acids,including the serine which forms part of the predicted catalytic centerof serine proteases. Chen, C., and Cleary, P., “Complete nucleotidesequence of the streptococcal C5a peptidase gene of Streptococcuspyogenes,” J. Biol. Chem., 265:3161-3167 (1990). An EcoRV site wascreated at the point of deletion. DNA which overlaps the deletion wassequenced to confirm the boundaries of the deletion.

The plasmid pG::ΔscpA1.1, which contains the deletion, was transformedinto E. coli ER1821. Colonies were selected for ErmR and then screenedfor the appropriate scpA deletion using miniprep plasmid DNA restrictedby EcoRI. The precise boundaries of the deletion were confirmed by DNAsequencing. Plasmid pG::ΔscpA1.1 was electroporated into strain CS101Smas described above, then integrants were selected by grown on Erm at 39°C. Integration of the plasmid into the chromosome of the M49 strainCS101sm using high temperature selection. The insertion location wasconfirmed by PCR. Growth of CS101Sm (pG::ΔscpA1.1) at low temperaturewithout erythromycin selection resulted in high frequency segregation ofErmS revertants which have lost the plasmid by random deletion event orby excision due to recombination between the duplicated scpA sequencescreated by the insertion. Two deletion mutants were identified, MJ2-5and MJ3-15, and were studied further. The chromosomal deletion leftbehind by recombinational excision of plasmid pG::ΔscpA1.1 was definedby PCR and Southern hybridization to EcoRV digested DNA.

e) In vitro effects of mutations on SCP. The impact of insertions anddeletions on the expression of SCP antigen and peptidase activity wasassessed by Western blot and PMNs adherence assays. Streptococci wereincubated in 100 ml THY at 37° C. overnight. The culture pellet waswashed two times in 5 ml cold 0.2 M NaAcetate (pH 5.2), then suspendedin 1 ml TE-sucrose buffer (20% sucrose 10 mM Tris, 1 mM EDTA, pH 7.0)and 40 μl mutanolysin. The mixture was rotated at 37° C. for 2 hr, thencentrifuged 5 mm at 4500 rpm. Supernatants contained protease inhibitor,100 mM phenylmethyl sulfonyl fluoride (PMSF). Electrophoresis andWestern blotting methods were performed as described in Laemmli, U. K.,“Cleavage of structural proteins during the assembly of the head ofbacteriophage T4,” Nature 227:680-685 (1970). The primary antiserum usedto detect SCP protein on Western and colony blots was prepared byimmunization of a rabbit with purified recombinant SCP protein. Bindingwas detected by anti-rabbit antibody alkaline phosphatase conjugate.

C5a peptidase activity was measured using a PMN adherence assay. Booth,S. A. et al., “Dapsone suppresses integrin-mediated neutrophil adherencefunction,” J. Invest. Dermatol. 98:135-140 (1992). After incubation ofC5a (Sigma, St. Louis, Mo.) with streptococcal extracts or purifiedprotease, residual C5a can activate PMNs to become adherent to BSAcoated wells. First, microtiter wells were coated with 0.5% BSA in PBSand incubated for 1 hr at 37° C. Human PMNs were isolated bycentrifugation in Ficoll® Hypaque™ solution (Sigma, St. Louis, Mo.). 40μl of intact streptococci or protein extracts were incubated with 20 μlof 5 μM C5a in 340 μl of PBS with 1% glucose and 0.1% CaCl₂ at 37° C.for 45 min. BSA-coated wells were washed with PBS, and resuspended PMNsand residual C5a were added to wells. The mixture was incubated for 45min at 37° C. in 7% CO₂. Finally, wells were washed to removenonadherent PMNs. Adherent PMNs were stained with crystal violet and theOD_(570nm) was read in an ELISA reader. The optical density isproportional to the amount of residual C5a or inversely proportional tothe amount of SCP activity.

Mutanolysin extracts of cell surface proteins from parent and mutantcultures were analyzed by Western blot using SCPA specific serum.Mutants were confirmed to lack SCPA. Extracts of SCPA⁻ mutants AK1.4 andMJ3-15 did not react with anti-SCPA serum. SCPA proteins of the expectedsize were observed in extracts from the wild-type strains CS101 andUAB200. Failure of mutant strains AK1.4 and MJ3-15 to produce C5apeptidase activity was verified by comparing their capacity to destroyrhC5a. Exposure of isolated PMNs to rhC5a induced them to becomeadherent to BSA coated microtiter wells. Incubation with streptococci orpurified SCPA specifically cleaved rhC5a and altered its potential toactivate PMNs. PMNs that responded to residual rhC5a and bound to BSAcoated wells, were stained, then measured spectrophotometrically.Incubation of rhC5a with parent cultures UAB200 and CS101 destroyedrhC5a, which inhibited PMN adherence by 58.8% and 54.5%, respectively.In contrast SCPA⁻ mutants, AK1.4 and MJ3-15, did not alter rhC5a oradherence of PMNs to BSA coated wells (Table 1). This experimentconfirmed the Western blots and demonstrated that SCPA⁻ cultures lackother proteases which might degrade rhC5a.

TABLE 1 Phagocytosis assay and PMN adherence assay of wild-type andmutant strains Colony forming units Percent inhibition of (cfu)/ml Foldincrease C5a induced PMN Strain Description Time = 0 h Time = 3 h incfu/ml adherence* UAB200 M6⁺, SCPA⁺ 1.8 × 10³ 7.2 × 10⁴ 40 58.8 AK1.4M6⁺, SCPA⁻ 1.2 × 10³ 4.5 × 10⁴ 37.5 0 CS101 M49⁺, SCPA⁺ 1.0 × 10⁴ 4.9 ×10⁵ 49 54.5 MJ3-15 M49⁺, SCPA⁻ 1.5 × 10⁴ 2.1 × 10⁵ 14 0 *Percentinhibition = [(OD_(570nm) of PMNs activated by C5a alone − OD_(570nm)PMNs activated by C5a preincubated with bacteria/OD_(570nm) of PMNsactivated by C5a alone)] × 100%.

Although M protein expression was not expected to be influenced bymutations in scpA, assays were performed to assess whether SCPA⁻ mutantstreptococci still expressed M protein and had the ability to resistphagocytosis. Growth of streptococci in fresh human blood during 3 hoursincubation is indicative of antiphagocytic M protein on their surface.R. C. Lancefield, “Differentiation of Group A Streptococci with a CommonR Antigen into Three Serological Types, with Special Reference toBactericidal Test,” J. Exp. Med., 106, pp. 525-685 (1957). As expected,parent streptococci UAB200 and CS101 increased 40 and 49 fold,respectively (Table 1). The M⁺ SCPA⁻ cultures, strains AK1.4 and MJ3-15,increased 37.5 and 14-fold, respectively, confirming that scpA mutationshad little effect on M protein expression or resistance to phagocytosisin whole human blood. The somewhat poorer growth of both mutant strainsin rotated blood was reproducible and unexpected. The growth rates ofmutant and parent cultures in human plasma were indistinguishable. It ispossible that inactivation of SCPA allowed C5a to accumulate in rotatedblood which in turn activated PMNs. Activated PMNs are more phagocyticand better able to kill M⁺ streptococci. Surface protein extractscontain M6 and M49 antigen when analyzed by Western blot using anti-M49and anti-M6 antisera, confirming that mutations in SCPA did not alter Mprotein expression.

EXAMPLE 2 SCP Delays Recruitment of Phagocytes and Clearance ofStreptococci from Subdermal Sites of Infection

In order to verify that SCP was responsible for the inactivation of C5a,the insertion and deletion mutants of scpA49 were constructed asdescribed in Example 1 above, and tested for activity. When insertionsor deletions were introduced into scpA49, the variant SCP was not ableto destroy C5a-activated adherence of PMNs to microtiter plates.

The impact of mutations in scpA49 on virulence was tested using ananimal model where streptococci remained localized, and where the influxof inflammatory cells could be analyzed. To test the hypothesis that SCPfunctions very early to retard initial clearance of the organism, thefate of SCP⁺ and SCP⁻ streptococci just 4 hours after inoculation ofconnective tissue air sacs was compared. Moreover, the dissemination ofstreptococci to lymph nodes and spleens after this short period ofinfection was also assessed.

CD1 male outbred mice (25 g) obtained from Charles River BreedingLaboratory, Wilmington, Mass. were used for all experiments. Aconnective tissue air sac was generated by injecting 0.9 ml of air and0.1 ml group A streptococci diluted in PBS with a 25-gauge needle underthe skin on the back of the mouse. In some experiments the SCP⁺CS101::pG⁺host5 was used as a positive control. In other experimentsstrain CS101Sm was used as the positive control. Mice were euthanized bycervical dislocation 4 hours after infection. Where indicated, all fouringuinal lymph nodes, spleen and air sac were dissected from the animalsand homogenized in PBS. Tissue suspensions were assayed for viablecolony forming unit (CFU) on blood agar plates containing 1 μg/mlerythromycin or 200 μg/ml streptomycin.

In a preliminary experiment air sacs were fixed on slides, stained withWright's stain and examined microscopically. Although counts ofgranulocytes by this method were unreliable, there appeared to besignificantly fewer residual SCP⁻ than wild-type streptococci in fixedtissue. Additional experiments were performed in an attempt to measurethis difference. Dispersed cell populations of air sacs were prepared bygrinding the air sac in PBS and passing them through Nylon monofilamentmesh (TETKO Co. New York).

The cells were pelleted by centrifugation 5 min at 300×g and resuspendedat 5×10⁶/ml in FACS buffer (Hank's balanced salt solution without phenolred, 0.1% NaN₃, 1.0% BSA fraction V). Cells (1.0×10⁶) were staineddirectly with 1 μg FITC anti-mouse Mac-1 or indirectly with 1 μg Biotinconjugated anti-mouse Gr-1 followed by 1 μg Streptavidin labelled withfluorescene or FITC. Monoclonal antibodies, Mac-1 and Gr-1, wereobtained from Pharmingen, Inc. Calif. Labeled cells were fixed in 1.0%paraformaldehyde. Fluorescence profiles were generated using a FAC-Scanflowcytometer and Consort 32 software (Becton Dickinson). Mouse PMNswere purified from whole blood by Ficoll Hypaque density gradientcentrifugation and used as a standard to defined PMNs in mixedpopulations. For measurement of specifically labeled cells, the meanfluorescence for each antibody marker was determined and gates were setto reflect intensely labeled cells. Controls included unstained cells,and cells exposed to only streptavidin FITC.

Two experiments were performed. The first compared the scpA49 insertionmutant M16 to its SCP⁺ parent culture, strain CS101. The second comparedthe scpA49 deletion mutant MJ3-15, to its parent, strain CS101Sm. (Table2) In both experiments homogenized air sacs from mice inoculated withSCP⁻ streptococci contained fewer numbers of streptococci after 4 hoursthan air sacs inoculated with wild-type streptococci. The firstexperiment showed a two-fold reduction and the second showed a four-foldreduction. These differences were statistically significant at P<0.05and P<0.001, respectively, using an Unpaired t-test. It was alsoobserved that wild-type SCP⁺ streptococci were found in spleenhomogenates from 7 of 8 mice and 6 of 8 mice; whereas, the SCP⁻ mutantswere rarely found in the spleen. The opposite was true for lymph nodehomogenates. Nodes from 10 of 16 mice infected with SCP⁻ streptococciharbored viable streptococci; whereas, only 4 of 16 nodes from miceinfected with wild-type streptococci contained viable bacteria. Thisdifference was determined to be statistically significant at P<0.05using the Fisher's exact test.

TABLE 2 Distribution of SCP⁺ and SCP⁻ streptococci 4 hours after air sacinfection No. of positive cultures No. of lymph Homogenized StrainsMice^(a) spleen^(b) node Air Sac^(c) CS101pG (SCP⁺) 8 7 2 1.3 × 10⁸ ±2.2 × 10⁷ M16 (SCP⁻) 8 0 5 6.0 × 10⁷ ± 1.3 × 10⁷ CS101Sm (SCP⁺) 8 6 21.6 × 10⁸ ± 2.6 × 10⁷ MJ3-15 (SCP⁻) 8 1 5 3.7 × 10⁷ ± 1.5 × 10⁷ ^(a)Eachmouse was inoculated with 3 × 10⁸ CFU of stationary phase streptococci.^(b)Difference in the frequency of isolation of SCP⁺ streptococci fromspleens relative to SCP⁻ streptococci was statistically significant (P <0.05) for each experiment by the Fisher's exact test. ^(c)Differences inCFU isolated from homogenized air sacs (means ± SEMs) were significant,strains CS101pG (SCP⁺) and M16 (SCP⁻) and MJ3-15 (SCP⁻) (P < 0.001) foreach experiment by unpaired t test.

The more rapid clearance of streptococci from air sacs resulted frommore intense recruitment of PMNs. The total cell population, thepercentage of Mac-1 positive granulocytes (Springer, G. et al.,“Mac-1:macrophage differentiation antigen identified by monoclonalantibody,” Eur. J. Immunol. 9:301-306 (1979)), and the percentage ofGr-1 positive PMN (Brummer, E. et al., “Immunological activation ofpolymorphonuclear neutrophils for fungal killing: studies with murinecells and blastomyces dermatitidis in vitro,” J. Leuko. Bio. 36:505-520(1984)) in air sacs were compared by single color FACS analysis. Clark,J. M., “A new method for quantitation of cell-mediated immunity in themouse,” J. Reticuloendothel. Soc. 25:255-267 (1979). Briefly, in a FACSanalysis, individual cells in suspension are labelled with specificfluorescent monoantibodies. Aliquots of labelled cells are injected intoa FAC-Scan flowcytometer or fluorescent cell sorter which counts cellsbased on their unique fluorescence.

Air sacs infected with the SCP⁻ deletion mutant contained twice as manyinflammatory cells as those inoculated with SCP⁺ streptococci (FIG. 4).A hundred-fold increase in the inoculum size did not alter thisdifference. Air sacs infected with 1×10⁶ SCP⁻ cells, strain MJ3-15,contained three times more Gr-1 positive cells than those inoculatedwith the SCP⁺ culture. In airs sacs inoculated with SCP⁺ streptococciapproximately 6% of the cells were PMNs and 21% were other kinds ofMac-1⁺granulocytes, including PMNs. In contrast, air sacs inoculatedwith SCP⁻ streptococci contained predominately PMNs. Gr-1 positive cellswere equal to or greater than the number of Mac-1 positive cells. Flowcytometer gates were set to measure only high staining granulocytes. Theremaining 70-80% of cells not stained with either antibody were likelyeither low staining granulocytes, red blood cells or lymphocytes. Largenumbers of lymphocytes were observed microscopically in Wrights stainedair sac preparations.

SCP⁺ colonies of streptococci that emerged from spleen homogenates werehighly encapsulated, resembling water drops. In contrast the few SCPcolonies arising from lymph nodes, were more like the inoculum. Theywere mixtures of non-mucoid and moderately mucoid colonies. These datasuggest that M⁺SCP⁺encapsulated streptococci can adapt, multiply andinvade the bloodstream within 4 hours after infection. The basis fordifferential trafficking of mutant and wild-type streptococci may be dueto the more vigorous influx of phagocytic cells in response to SCP⁻bacteria. Macrophages and/or skin dendritic cells may more rapidlyengulfed SCP streptococci and delivered them to lymph nodes. Reductionof mutant streptococci relative to wild-type is an unexpected finding,because SCP⁻ streptococci are M⁺ and resistant to phagocytosis by humanneutrophils in vitro.

EXAMPLE 3 SCP Is Required for Colonization of the Mouse Nasopharynx

Mice were inoculated intranasally to evaluate the relative capacity ofwild-type (SCP⁺) and SCP⁻ streptococci to colonize the nasopharynx.Streptomycin resistant M49 strain CS101 and deletion mutant MJ3-15 wereused in these experiments. Cultures were not mouse passed in order toavoid selection of variants that might be uniquely mouse virulent, butno longer depend on M protein and/or SCP for persistence in the animal.

Sixteen hour cultures of challenge streptococcal strains (1×10^(8-9×10)⁸ CFU), grown in Todd-Hewitt broth containing 20% normal rabbit serumand resuspended in 10 μl of PBS, were administered intranasally to 25 gfemale CD1 (Charles River Breeding Laboratories, Inc., Wilmington,Mass.) or BALB/c mice (Sasco, Omaha Nebr.). Viable counts weredetermined by plating dilutions of cultures on blood agar plates. Throatswabs were taken daily from anesthetized mice for 6 to 10 days afterinoculation and streaked onto blood agar plates containing 200 ug/mlstreptomycin. After overnight incubation at 37° C., the number ofβ-hemolytic colonies on plates were counted. All challenge strains weremarked by streptomycin resistance to distinguish them from β-hemolyticbacteria which may be persist in the normal flora. Throat swabs werecultured on blood agar containing streptomycin. The presence of oneβ-hemolytic colony was taken as a positive culture.

CD1 outbred mice were intranasally inoculated with 2×10⁸ stationaryphase CFU. The nasopharynxes of anesthetized mice were swabbed daily for8-10 days and streaked on blood agar containing streptomycin.Differences between SCP⁺ and SCP⁻ were evident by day 1, however,statistically significant differences were not observed until days 3 and4 (FIG. 5). By day four 9/18 mice infected with M⁺SCP⁺ streptococciproduced positive throat cultures, whereas only 2/18 mice infected withM⁺SCP⁻ strain retained streptococci in their throats. Four of 18 micedied from infection with SCP⁺ streptococci. None of the mice followinginfection with SCP⁻ bacteria succumbed to the infection. The numbers ofcolonies on the blood agar plates were also consistent with more rapidclearance of SCP⁻ streptococci. For example, on the third day culturesfrom seven mice contained >100 SCP+CFU, whereas, only one mouseinoculated SCP⁻ streptococci contained >100 CFU.

Because M49 streptococci are more often associated with skin infectionsthe above experiments were repeated with an M6 strain, a serotype moreoften associated with throat infections. An insertion mutant, strainAK1.4, was constructed using the M6 strain UAB200 and the strategypreviously described in Example 1. Strain AK1.4 was also cleared morerapidly than the wild-type M6 culture from the nasopharynx. (FIG. 6) Theabove experiments confirm that group A streptococci are dependent uponSCP for persistence in the mouse nasopharynx. All SCP⁻ variants used inthe above experiments were M⁺, i.e. they resisted phagocytosis by freshhuman blood. Yet, they were cleared from the nasopharyngeal mucosa.

EXAMPLE 4 Intranasal Immunization of Mice with Purified RecombinantSCPA49 Blocks Colonization Following Intranasal Challenge

a) Construction of recombinant vaccine ΔSCPA49 encoding Thr⁶³ throughHis¹⁰³¹ (FIGS. 2 and 7).

A PCR fragment which corresponds to a truncated form of the scpA49 genewas cloned from CS101 M49 group A streptococci (ΔSCPA49). This fragmentwas amplified by PCR using a forward primer beginning at nucleotide 1033and a reverse primer beginning at nucleotide 3941 (numberingcorresponding to that of Chen, C., and Cleary, P., “Complete nucleotidesequence of the streptococcal C5a peptidase gene of Streptococcuspyogenes,” J. Biol. Chem., 265:3161-3167 (1990)). The fragment wasligated to the thrombin binding site of glutathione transferase gene onthe pGex-4T-1 high expression vector from Pharmacia Inc. The plasmidcontaining the recombinant scpA fragment, designated pJC6, has beendeposited in the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, under the provision of the BudapestTreaty, and assigned ATCC accession number 98225.

The ΔSCPA49, a 2908 bp fragment of scpA49, was amplified by PCR using anscpA49 forward primer containing a BamHI recognition sequence(5′-CCCCCCGGATCCACCAAAACCCCACAAACTC-3′) (SEQ ID NO:8) and an scpAreverse primer (5′-GAGTGGCCCTCCAATAGC-3′) (SEQ ID NO:9). Sequences whichcode for the signal peptide and membrane anchor regions of the SCPAprotein were deleted from the resulting PCR product. PCR products weredigested with BamHI and ligated to BamHI and SmaI restriction sites inthe thrombin recognition site of the glutathione S-transferase gene onthe pGEX-4T-1 high expression vector from Pharmacia Inc. (Piscataway,N.J.). The recombinant plasmid was transformed into E. coli DH5α. TheΔSCPA49 fusion protein from one transformant, E. coli (pJC6), waspurified by affinity chromatography on a glutathione Sepharose® 4Bcolumn. The transferase-SCP fusion protein from one E. coli clone wasexpressed and purified by affinity chromatography on a glutathioneSepharose® 4b column. All methods are described by the manufacturer(Pharmacia). The ΔSCPA49 was cleaved from the hybrid protein by thrombindigestion. The thrombin was removed from eluted SCP by chromatography ona benzamidine 8Sepharose® 6B column (Pharmacia). Following digestionwith thrombin, thrombin was removed by chromatography on a benzamidineSepharose® 6B column. Methods of expression and purification aredescribed by the manufacturer. The affinity purified protein wasconfirmed to be pure ΔSCPA49 by SDS-PAGE and by Western blot. Thisaffinity purified, truncated ΔSCPA49 protein lacked peptidase activitywhen tested by the PMN adherence assay (described in Example 1 above).Hyperimmune antiserum, directed against purified ΔSCPA49 was prepared inrabbits.

b) Immunization and challenge protocol. Four week old, outbred, CD 1female mice were immunized by administration of 20 μg of affinitypurified ΔSCPA49 in 10 μl PBS into each nostril. Mice were immunized 3times on alternating days and boosted again three weeks after the thirdimmunization. After two weeks rest, mice were again boosted. D. Bessenet al., “Influence of Intranasal Immunization with Synthetic PeptidesCorresponding to Conserved Epitopes of M Protein on Mucosal Colonizationby Group A Streptococci,” Infect. Immun., 56, pp. 2666-2672 (1988).Control mice received only PBS. Prior to infection, all mice which wereimmunized with ΔSCPA49 protein were determined by ELISA to have hightiters of antibodies against ΔSCPA49 antigen in their serum and saliva.Group A streptococci, strain CS101 (2.0×10⁸ CFU), CS210 (3.6×10⁸ CFU),CS463 (7.8×10⁸ CFU), 90-131(3.4×10⁸ CFU), and UAB200 (9.6×10⁸ CFU) wereused to intranasally challenge the mice 7 days after the last vaccinebooster. Animal studies were performed according to National Institutesof Health guidelines.

c) Sample collection and ELISA. Blood and saliva samples were collectedfrom anesthetized mice after immunization. All sera were tested for thepresence of SCPA49 antibodies by ELISA, as previously described. S. P.O'Connor et al., “The Human Antibody Response to Streptococcal C5aPeptidase,” J. Infect. Dis., 163, pp. 109-116 (1990). Purified SCPA49protein was bound to microtiter wells by addition of 500ng of purifiedprotein in 0.05M bicarbonate buffer (pH 9.6). After overnight incubationat 4° C. the wells were washed, then blocked with 0.5% BSA in PBS for 1hour. Salivation was stimulated in mice by injection of 100 μl of a 0.1%pilocarpine (Sigma) solution subcutaneously. Saliva samples werecollected and spun at 14,000 rpm for 5 min in an Eppendorf®microcentrifuge. The supernatants were tested for the presence ofsecretory IgA against ΔSCPA49 protein by ELISA. ELISA titers representthe highest dilution of individual serum and saliva which had an OD₄₀₅≧0.1.

d) Evaluation of Antibody Response to ΔSCPA49

The immunogenicity of the subunit ΔSCPA49 vaccine was evaluated. Rabbitswere immunized with purified ΔSCPA49. The rabbits developed high levelsof antibodies against ΔSCPA49 protein as determined by ELISA. Althoughthe purified ΔSCPA49 immunogen lacked functional activity, hyperimmunerabbit antiserum could neutralize the peptidase activity of purifiedwild-type SCPA49 enzyme in vitro. Moreover, undiluted rabbit antiserumagainst ΔSCPA49 protein was able to neutralize C5a peptidase activityassociated with different serotypes (FIG. 8). C5a peptidase activityassociated with intact M1, M6 and M12 streptococci was inhibited by thisantiserum, confirming that antibody against ΔSCPA49 protein lacksserotype specificity.

Also, serum and saliva samples were obtained from ten immunized and tencontrol mice to assess the immunogenicity of ΔSCPA49 protein whenadministered via the intranasal route without adjuvants. Mice which wereimmunized with purified ΔSCPA49 protein developed high titers ofΔSCPA49-specific IgG in their sera, compared to control mice immunizedwith PBS (FIG. 9). Titers of serum IgG directed against ΔSCPA49 rangedfrom 1:10,240 to 1:20,480. In contrast, ΔSCPA49-specific IgG titer ofcontrol mice was not detectable in sera. Mice immunized with purifiedΔSCPA49 protein also showed a significant increase in ΔSCPA49-specificsalivary sIgA relative to control mice. Specific sIgA titers in salivaof immunized mice were greater than 1:16. In contrast, sIgA directedagainst ΔSCPA49 in the saliva of control mice was not detectable. Therelative concentration of IgG and sIgA in serum diluted 1/2560 andsaliva diluted 1/2, respectively, are shown in FIG. 9. These resultsdemonstrate that purified ΔSCPA49 protein is an effective immunogen forthe induction of specific systemic and secretory antibody responses inmice when administered intranasally.

e) Impact of Vaccine ΔSCPA49 on Clearance of Streptococci from InfectedMice.

Experiments were performed to determine whether immunization with theC5a peptidase would enhance clearance of streptococci from thenasopharynx. Both hyperimmune rabbit and human sera that contain highlevels of anti-SCPA antibody can neutralize SCPA activity in vitro. S.P. O'Connor et al., “The Human Antibody Response to Streptococcal C5aPeptidase,” J. Infect. Dis., 163, pp. 109-116 (1990). The fact that SCPAsignificantly facilitates colonization of the oral mucosa suggests thatimmunization of mice with purified ΔSCPA49 could reduce the capacity ofstreptococci to colonize the nasopharynx. Mice were immunizedintranasally with affinity purified, genetically inactivated SCPA totest this possibility. The truncated protein, ΔSCPA49, was administeredintranasally without adjuvants or carriers. Pharyngeal colonization ofvaccinated mice by wild-type M⁺ SCPA⁺ streptococci differedsignificantly from those immunized with PBS in three independentexperiments using mice vaccinated with two different preparations ofpurified ΔSCPA49 protein (Tables 3 and 4; FIG. 10). Only one of 13 miceimmunized with ΔSCPA49 protein was culture positive for streptococci tendays after inoculation (Table 4; FIG. 10). In contrast, 30-58% of thenon-vaccinated controls remained culture positive for six days, and somewere still positive ten days after infection. The numbers ofβ-hemolytic, streptomycin resistant colonies on blood agar plates alsoshowed a significant difference between ΔSCPA49 vaccinated and controlmice. Different sets of immunized mice cleared serotype M49 streptococcisignificantly more rapidly from their nasopharynx than non-immunizedcontrol.

TABLE 3 Throat cultures for streptococci after intranasal challenge ofmice vaccinated intranasally with PBS or SCP expressed in E. coli DH5α(CFU after vaccine) Days after challenge Mice 1 2 3 4 5 6 7 8 9 10PBSCT-II  1 0 0 0 0 0 0 0 0 0 0  2 3 0 0 0 0 0 0 0 0 0  3 77 >200 150 411 3 0 51 97 53  4 9 >200 >200 3 11 3 0 0 0 0  5 0 0 0 0 0 0 0 0 0 0  64 6 45 47 3 >200 29 >200 83 70  7 15 194 >200 9 172 10 5 3 0 0  8 0 0 00 0 0 0 0 0 0  9 0 32 4 4 0 0 0 0 0 0 10 2 0 0 0 0 0 0 0 0 0 11 3 0 0 00 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 13 127 4 0 0 0 0 0 0 0 0 No. of 8 6 55 4 4 2 3 2 2 positive SCPAD-II  1 0 0 0 0 0 0 0 0 0 0  2 0 0 0 0 0 0 00 0 0  3 0 0 0 0 0 0 0 0 0 0  4 0 0 0 0 0 0 0 0 0 0  5 35 0 0 0 0 0 0 00 0  6 0 0 0 0 0 0 0 0 0 0  7 0 0 0 0 0 0 0 0 0 0  8 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 11 0 0 0 21 0 0 0 0 0 0 120 0 0 0 0 0 0 0 0 0 13 0 0 0 0 0 0 0 0 0 0 No. of 1 0 0 1 0 0 0 0 0 0positive

TABLE 4 Throat cultures for streptococci after intranasal challenge ofmice vaccinated intranasally with PBS or SCP expressed in E. coli DH5α(CFU after vaccine) Days after challenge Mice* 1 2 3 4 5 6 7 8 9 10PBSCT-I  1 112 143 85 16 0 0 0 0 0 0  2 127 27 18 89 3 7 7 7 70 3 3 >200 >200 >200 >200 >200 >200 >200 108 >200 66  4 31 200 4 2 0 0 0 00 0  5 4 0 0 3 3 0 0 0 0 0  6 0 0 0 0 0 0 0 0 0 0  7 >200 >200 120 12591 145 >200 >200 >200 166  8 2 0 0 0 0 0 0 0 0 0  9 0 0 0 0 0 0 0 0 0 010 0 0 0 0 0 0 0 0 0 0 11 37 >200 194 16 >200 47 >200 101 >200 >200 No.of positive 8 6 6 7 5 4 4 4 4 4 SCPAD-I  1 6 0 0 0 0 0 0 0 0 0  2 105 410 0 0 0 0 0 0 0  3 0 0 0 0 0 0 0 0 0 0  4 2 0 0 0 0 0 0 0 0 0  5 2 0 0 00 0 0 0 0 0  6 9 0 11 0 0 0 0 0 0 0  7 0 0 0 0 0 0 0 0 0 0  8 26 0 0 0 00 0 0 0 0  9 0 19 0 0 5 57 0 0 21 91 10 0 0 0 0 0 0 0 0 0 0 11 7 0 0 0 00 0 0 0 0 No. of positive 7 2 1 0 1 1 0 0 1 1 *Mice were inoculatedtwice, because the dose of bacteria was too low at first timeinoculation.

Last, it was examined whether SCP of one serotype would vaccinateanimals against infection from other serotypes. There are more than 80different serotypes of group A streptococci. An effective vaccine shouldprevent infection to more than one streptococcal serotype.Cross-protection was produced against colonization by the streptococcalOF⁺ serotypes M2 and M11 and the OF⁻ serotypes M1 and M6. The fact thatrabbit serum directed against ΔSCPA49 protein from serotype M49streptococci neutralized peptidase activity associated with severalserotypes suggested that intranasal immunization with a single subunitvaccine might reduce or eliminate pharyngeal colonization by thoseserotypes. To explore this possibility four groups of twenty mice wereimmunized by intranasal inoculation with affinity purified ΔSCPA49protein as described above. Control mice received PBS. Prior to beingchallenged with streptococci, serum and saliva samples from randomlychosen, immunized and control mice were assayed for anti-SCPA antibody.All immunized mice tested had developed a strong serum and measurablesalivary antibody response. Pharyngeal colonization of mice immunizedwith ΔSCPA49 protein by strains of all four serotypes was reducedrelative to non-immunized controls. Differences were most significant ondays 3 and 5 after inoculation (Table 5).

TABLE 5 Immune protectivity is serotype independent Day 3 after Day 5after inoculation inoculation Nonimmune Immune Nonimmune Immune(+/total) % (+/total) % (+/total) % (+/total) % M2 10/19 52.6  2/19*10.5 3/19 15.8 1/19 5.2 M11 17/20 85 11/20* 55 8/20 40 2/20* 10 M1 16/1984.2 11/19 57.9 7/19 37 2/19* 10.5 M6 14/20 70 12/19 63.2 8/20 40 4/1921.1 + means culture positive mice. *Differences between immunized andnon-immunized mice are statistically significant (P < 0.05). P valueswere calculated by x² analysis.

Statistically significant differences were observed between immunizedand control mice inoculated with serotype M2, M11 and M1 strains.However, the OF⁺ serotypes M2 and M11 were more efficiently eliminatedby immunized mice than were the OF⁻ strains, M1 and M6. M1 streptococcalcolonization of immunized mice was significantly reduced relative tocontrol mice. Only 10.5% of the immunized mice were culture positive byday 5 post-infection. In contrast, 37% of the control mice were culturepositive with this strain. Although immunized mice appeared to clear M6streptococci more rapidly, the differences were not statisticallysignificant. As in previous experiments the number of β-hemolyticstreptococcal colonies on blood agar plates were significantly fewer insamples taken from vaccinated mice than those taken from controlanimals. Thus, the ΔSCPA 49 protein was an effective vaccine thatprovided cross-protection against other streptococcal serotypes.

EXAMPLE 5 Site-directed Mutagenesis of SCPA49

Group A streptococcal serotypes can be divided into two major groups,OF⁺ and OF⁻ strains. The latter are more often associated with rheumaticfever and toxic shock, whereas OF⁺ strains are a common cause ofimpetigo and acute glomerulonephritis. Although the SCPA proteins ofthese groups are 95-98% identical, it is possible that the immuneresponse to them may be somewhat different. This concern promptedefforts to develop defined variant SCPAs from an M1 OF⁻ strain and froman M49 OF⁺ strain in parallel. Amino acids that are required forcatalytic activity were replaced with those expected to inactivate theenzyme (FIG. 1). The N and C-terminal amino acid boundaries of SCPA49,expressed the pGEX-4T-1 subclones, were Asn³² and His¹¹³⁹, respectively(FIGS. 1 and 8). Ser⁵¹² (SCPA49S512A), Asn²⁹⁵ (SCPA49N295A) and Asp¹³⁰(SCPA49D130A) in the SCPA49 protein were replaced with Ala, and Asn²⁹⁵(SCPA49N295R) was replaced by Arg (Deborah Stafslien, M. S. Thesis,University of Minnesota).

The method used to introduce mutations into the scpA49 gene fromStreptococcus strain CS101 was the “megaprimer” method of site-directedmutagenesis. Barik, S., “Site directed mutagenesis in vitro megaprimerPCR,” In: Methods in Molecular Biology, Vol. 57: In Vitro MutagenesisProtocols, Humana Press, Inc. Totowa, N.J. (1996). The serine mutationwas introduced using primers scpFor940(5′-CCCCCCGGATCCAATACTGTGACAGAAGACACTCC-3′), SEQ ID NO:10, andscpmutrev1883 (5′-TTTCTGGAACTAGTATGTCTGCGCC-3′), SEQ ID NO:11, toamplify a 1450 bp double-stranded PCR product. This first PCR product, a“megaprimer,” was purified using the Qiagen Qiaquick Gel Extraction Kit,then used in a second asymmetrical PCR reaction to amplify the 3.3 kbscpA49 gene containing the desired mutation. Five cycles of denaturation(93° C., 1 min) and extension (72° C., 5 min) were carried out beforeaddition of the reverse primer scpRev4263,(3′-CCCCCCCTCGAGATGTAAACGATTTGTATCCTTGTCATTAG-3′) SEQ ID NO:12. Duringthe fifth cycle at 72° C., the reverse primer was added at aconcentration of 1 mM. The amplification was completed using 25 cyclesat 94° C. for 1 min, 58° C. for 2 min, and 72° C. for 2-3.5 minutes.Reactant concentrations were the same as described in the previoussection, except that a forward primer was not added and the megaprimerwas added at a concentration of 4-6 μg per 100 μl reaction. This processyielded variant protein SCPA49S512A (see Table 6 below).

The aspartate and asparagine variants were constructed in much the samefashion, using the reverse primers scpmutrev717(5′-CAGTGATTGATGCTGGTTTTGATAA- 3′) SEQ ID NO:13 and scpmutrev1214(5′-AGCTACTATCAGGACCAG- 3′) SEQ ID NO:14 to construct 311 bp and 805 bpmegaprimers, respectively. The primer scpmutrev717 was used to generatevariant protein SCPA49D130A, and primer scpmutrev1214 was used togenerate variant protein SCPA49N295A (see Table 6 below). AfterQiaquick™ purification, however, the megaprimer was treated with 0.1 Umung bean nuclease (per 4 μg DNA) and incubated at 30° C. for 10minutes. The nuclease was removed by phenol/chloroform extraction, andthe megaprimer recovered in the aqueous phase by ethanol precipitation.The pellet was resuspended in 80 μl sterile double distilled water, and37 μl of this was used in each 100 μl asymmetrical PCR reaction. Themutated gene was then cloned into pGEX 4T-1 as previously described.Sequencing of variants was performed using ³⁵S-labeled dATP and theSequenase™ kit (Stratagene®) or using automated fluorescent sequencingat the University of Minnesota Microchemical Facility.

TABLE 6 Amino acid sequence comparison of variant proteins Wild-type 127132 291 297 508 514 876 883 SCPA49 AVIDAG TSAGNDS LSGTSGT STLGSRF (SEQID NO:24) (SEQ ID NO:25) (SEQ ID NO:27) (SEQ ID NO:28) SCP S512A49AVIDAG TSAGNDS LSGTAGT STLGSRF (SEQ ID NO:24) (SEQ ID NO:25) (SEQ IDNO:26) (SEQ ID NO:28) SCP D130A49 AVIAAG TSAGNDS LSGTSGT STLGSRF (SEQ IDNO:29) (SEQ ID NO:25) (SEQ ID NO:27) (SEQ ID NO:28) SCP N295A49 AVIDAGTSAGADS LSGTSGT STLGSRF (SEQ ID NO:24) (SEQ ID NO:30) (SEQ ID NO:27)(SEQ ID NO:28)

The E. coli expression vector pGEX 4T-1 was used to overexpress variantSCPA as GST fusion proteins. Recombinant SCPA was purified according tothe protocol provided in the GST Gene Fusion System Handbook (Pharmacia)previous to this work. The SCPA protein antigen was purified by affinitychromatography as described above.

EXAMPLE 6 Construction of SCPA1 and SCPB Variants

The wild-type scpA1 gene was amplified by PCR from M1 serotype of S.pyogenes (strain 90-226) in the following manner. Primers were designedsuch that only a fragment of the complete gene would be expressed. Thisfragment corresponds to the start of the mature protein and terminatesjust before the cell wall associated domain residue Asn³² throughAsp¹⁰³⁸ (FIG. 2). The forward primer 5′-CCCCCCGAATTCATTACTGTGACAGAAGACACTCCTGC-3′ (SEQ ID NO:15) anneals starting at base number 940(numbering corresponding to that of Chen, C., and Cleary, P., “Completenucleotide sequence of the streptococcal C5a peptidase gene ofStreptococcus pyogenes,” J. Biol. Chem., 265:3161-3167 (1990). Theopposing, reverse PCR primer, 5′-CCCCCCGGATCCTTATTGTTCTGGTTTATTAGAGTGGCC-3′ (SEQ ID NO:16) anneals at base number 3954 just upstream of aregion of DNA repeats. This repeat region of the protein is predicted tobe the part that passes through, and then attaches to the peptidoglycanof the cell wall. The italicized region of each primer is additionalsequence that has been added to the S. pyogenes sequence to enable thecloning process. The underlined region of the forward primerincorporates a EcoRI restriction site, the underlined portion of thereverse primer a BamHI site. The reverse primer also incorporates a stopcodon (TAA) in frame of the gene that terminates translation.

The resultant PCR product corresponding to bases 940-3954 was clonedinto an intermediate vector pCR2.1 (Invitrogen™, Inc.) and transformedinto E. coli Top10F cells (Invitrogen™, Inc.). Plasmid DNA from anappropriate transformant was restricted with EcoRI and BamHI. The 3018base fragment, containing the fragment of scpA1, was gel purifiedfollowing standard procedures and ligated into the expression vectorpTrc99a (Pharmacia) restricted with the same enzymes. This ligation wastransformed into E. coli DH5α cells and a transformant was selected thatcontained the desired plasmid construction. The resultant plasmid placesthe PCR fragment of scpA1 behind a Shine-Dalgarno sequence and ATG startsite, and is under the transcriptional control of the trc Promoter, thatis inducible with the allolactose analogue IPTG.

Site-specific genetic variants of the wild-type scpA1 were constructedfollowing a procedure described by C. L. Fisher and G. K. Pei,“Modification of a PCR-based site-directed mutagenesis method,”BioTechniques, 23:570-574 (1997). The appropriate amino acid residueswithin SCPA1 important for protease activity were predicted by sequencecomparisons to the family of subtilisin-like serine proteases. Siezen,R. J., et al., “Homology modeling and protein engineering strategy ofsubtilases, the family of subtilisin-like serine proteinases,” ProteinEngineering, 4:719-737 (1991); Chen, C., and Cleary, P., “Completenucleotide sequence of the streptococcal C5a peptidase gene ofStreptococcus pyogenes,” J. Biol. Chem., 265:3161-3167 (1990). Threeresidues, conserved amongst this family, are involved in the formationof the active site. In SCPA1, these correspond to the Asp¹³⁰, His¹⁹³,and Ser⁵¹². Three sets of non-overlapping oligonucleotides were designedfor use in PCR to alter each one of these amino acid residues. Theseoligonucleotides were designed to amplify away from each other onopposite strands of DNA. In each set, the 5′ end of one of the primerswould contain the codon encoding one of these amino acids for mutationand this codon would be altered to encode an alanine. These three setsof primers are listed below; the codons that are changes are italicized.

D130A: Forward (SEQ ID NO:17) 5′-ATT GCT GCT GGT TTT GAT AAA AAT CAT GAAGCG-3′ GAT codon change to GCT corresponds to an aspartate to alanineamino acid change. Reverse (SEQ ID NO:18) 5′-CAC TGC AAC AAC AGT CCC-3′H193A: Forward (SEQ ID NO:19) 5′-GAG GCC GGC ACA CAC GTG-3′ CAC codonchange to GCC corresponds to a histidine to alanine amino acid change.Reverse (SEQ ID NO:20) 5′-TTG ATC GAC AGC GGT TTT ACC-3′ S512A: Forward(SEQ ID NO:21) 5′-ACT GCT ATG TCT GCT CCA TTA G-3′ ACT codon change toGCT corresponds to a serine to alanine amino acid change. Reverse (SEQID NO:22) 5′-TCC AGA AAG TTT GGC ATA CTT GTT GTT AGC C

These sets of PCR primers were used in three separate reactions. Thetemplate DNA was pLP605, which contained the wild-type scpA1 sequence.The PCR products were subsequently self-ligated and transformed into theE. coli strain Top10F′ Invitrogen™, Inc.). Transformants were screenedfor the appropriate size and restriction pattern. The sequence change inthe S512A variant destroys a unique SpeI restriction site so that thismutation could be identified directly by restriction analysis. Allpotential variants were confirmed by DNA sequencing. Subsequently, theD130A mutation was combined with the S512A mutation to form a doublevariant utilizing a unique PstI site between these two regions of theprotein. The final alteration was to change the antibiotic selectionfrom ampicillin to kanamycin by moving the variant scpA1 genes to apreviously altered pTRC99a vector (Pharmacia, Inc.) containing thekanamycin gene.

A variant of SCPB protein was constructed using the method describedabove for SCPA1 mutants. The wild-type SCPB gene was cloned from group Bstreptococcus 78-471 (Type IIa⁺).

EXAMPLE 7 Analysis of Variant Proteins

Proteins expressed from each of the variant constructs were analyzed bySDS polyacrylamide gel electrophoresis. The expected size of the proteinis 121 kD, however, the proline-rich cell wall spanning domain at thecarboxy terminus of the enzyme causes the protein to run slightly slowerduring SDS-PAGE. Therefore the apparent molecular weight is 130 kD whendetermined by SDS-PAGE. Since active SCP could be harmful to the host,it was important that the variant proteins lacked enzymatic activity.Two properties of the variant proteins were evaluated. The specificactivities of the wild-type and variant proteins as determined by PMNadherence assay are compared in Table 7. These experiments indicatedthat the substituted amino acids reduced enzymatic activity by greaterthan 90%.

TABLE 7 PMN adherence assay determination of variant protease activityProtein Activity (U/mg * 10⁻³) Wild-type 170 SCPA49D130A <20 SCPA49N295A<20 SCPA49S512A <20

The variant proteins were also compared to the wild-type protein fortheir capacity to bind antibody directed against the wild-type enzyme.Competitive ELISA assays were used for this purpose. Competitive ELISAsmeasured the inhibition of antibody binding to immobilized antigen bysoluble antigen. A constant amount of wild-type antigen was bound towells of the microtiter plate. A constant amount of antibody is added atthe same time with varying amounts of soluble competitive antigen. Theslope of the percent inhibition versus antigen concentration curvesestimate the relative binding affinity of the soluble antigen forantibody. While the binding constants cannot be calculated withoutknowing the exact concentration of anti-SCPA in the antiserum, therelative binding affinities of several proteins were compared (FIG. 11).Since the slopes of the percent inhibition versus concentration curvesare the same for the wild-type and variant proteins, it was concludedthat amino acid substitution did not alter the ability of antibody tobind to the variant proteins.

Recombinant SCPA1, SCPA49 and SCPB proteins were also determined to bindequally well to anti-SCP antibody (FIG. 12). In this experiment theplate antigen was SCPA49 and the antibody was rabbit anti-SCPA49. Therelative affinities of this antibody for these antigens, indicated bythe slope of the curves is highly similar. These results demonstratethat SCPA protein from M49 OF⁺ and M1 OF⁻ group A Streptococci, and fromgroup B streptococci are equivalent with regard to antibody recognitionand may be used interchangeably in a vaccine preparation.

EXAMPLE 8 Subcutaneous (SQ) Administration of SCPA Antigen InducesProtection in Mice

All earlier protection studies were performed by administering affinitypurified SCPA49 protein intranasally without adjuvant. Intramuscular orSQ injection of antigens is historically a preferred, more acceptedmethod of vaccine delivery. Therefore, experiments were performed totest whether SQ injections of SCPA with MPL/alum induced a protectiveimmune response and whether that response reduced colonization when thechallenge strain of group A streptococcus differed in serotype from thesource of the SCPA vaccine. The capacity of immunized mice to clearstreptococci from the oral-nasal pharyngeal mucosa was evaluated bythroat culture or by sampling dissected nasal tissue. Representativethroat culture data are presented in Table 8.

TABLE 8 Subcutaneous vaccination of mice Percent Colonized^(c)SCPA-Immunized Vaccine^(a) Challenge Bacteria^(b) Control Mice MiceSCPA49S512A OF⁺M49 64% (3) 36% ΔSCPA49 OF⁺M49 64% (3) 20% ΔSCPA49 OF⁻M133% (5)  8% SCPA1S512A OF⁻M49 23% (5)  8% ^(a)Vaccines contained 10 μgof the indicated antigens mixed with adjuvants MPL and alum.Experimental groups each contained 13-20 mice. Control mice wereimmunized with tetanus toxoid mixed with the same adjuvant. ^(b)Micewere infected by intranasal inoculation. ^(c)Colonization was assessedby throat culture. The numbers in parentheses indicate the day on whichthe cultures were taken.

Mice immunized by SQ injection of each of the three different forms ofSCPA antigen induced moderate protection. Immunization with ASCPA49protected against both OF⁻ M1 and OF⁺ M49 strains. SCPA49S512A andSCPA1S512A were chosen for subsequent study.

Persistence of streptococci following intranasal challenge was alsoassessed by a more quantitative assay. This method involved sacrificinggroups of mice at different times following infection, and dissectingnasal tissue (NT), which was then assayed for viable streptococci (CFU).Standard amounts of NT were homogenized in buffer and the number ofCFU/mg tissue was determined by viable count.

Three groups of mice were immunized SQ with SCPA49S512A, SCPA1S512A ortetanus toxoid. All vaccines were mixed with MPL/Alum adjuvants asbefore. Mice received four injections of 5 μg protein antigen and thenchallenged two weeks after the last injection. Nasal tissue washarvested 16 hours after challenge with the OF⁺ M49 strain CS101. Thegeometric means of CFU/mg tissue are shown in Table 9.

TABLE 9 Geometric means of CFU/mg nasal tissue Vaccine Antigen 16hours^(a) Tetanus 5.71^(b) SCPA49S512A 2.27 SCPA1S512A 1.60 ^(a)The timeat which NT was taken following intranasal infection of mice. ^(b)Valuesare log values.

The number of streptococci associated with nasal tissue decreased withtime, as expected and the decrease was more rapid and complete in miceimmunized with SCPA antigen. All groups of mice that had been immunizedwith SCPA retained fewer streptococci than control mice. In thisexperiment immunization with SCPA1S512A was most effective and induced across-protective response, since the challenge strain CS101 is OF⁺ M49and the source of vaccine protein SCPA1S512A from an OF⁻ M1 strain.These results confirm that a single SCPA antigen can induce protectionagainst heterologous serotypes. Protection is afforded by antibody thatneutralizes peptidase activity on the bacterial surface. This increasesthe influx of phagocytes within a few hours from the time streptococciare deposited on mucosal tissue. Rapid clearance of streptococci byphagocytes is presumed to prevent subsequent multiplication andpersistence of the bacteria. Mice uniformly had serum IgG titers of1:32,000 or greater when assayed by ELISA, indicating that SQ injectionof SCPA antigen with adjuvant consistently induced a vigorous antibodyresponse.

EXAMPLE 9 C5a Peptidase from Group B Streptococci Is Nearly Identical inSequence to Those from M12 and M49 Group A Streptococci

The group B streptococci C5a peptidase (SCPB) gene was cloned, sequencedand compared to that from serotype group A streptococci M12 and M49. Theentire scpB gene was amplified by PCR using primers which correspond toportions of the scpA12 sequence using the method described above. TheSCPB gene encodes an open reading frame (ORF) of 3450 bp which specifiesa protein of 1150 amino acids with Mr of 126,237 da. The amino acidsequence of SCPB is shown in FIG. 2. Comparison of the scpB nucleotideand deduced amino acid sequence to those from M12 and M49 group Astreptococci showed high similarities, 98% and 97%, respectively. ScpBcontained a 50 bp deletion which overlapped two of the C-terminalrepeats, and had several other minor differences relative to scpA genes.Alignment of the sequences showed that scpA12 is actuallyphylogenetically closer to scpB than it is to scpA49. Thirty strains,representing serotypes III, IIIR, II, Ia/c, NT/c, NT/c/R1 carry a copyof scpB.

Recombinant SCP was expressed in E. coli using expression vector plasmidpGEX-4T-1 (ATCC accession number 98225) and was shown to be identical tothe enzyme extracted from the parental group B streptococcal strain78-471 (Type II a+b). Western blot analysis suggested the recombinantSCP is identical to the C5ase enzyme previously purified from group Bstreptococci.

All publications, patents and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the scope of the invention.

1. An isolated and purified enzymatically inactive peptide of peptide ofStreptococcal C5a peptidase (SCP), wherein the peptide comprises thecatalytic domain of the SCP, which elicits antibodies that neutralizethe peptidase activity of wild-type SCP.
 2. The peptide of claim 1wherein the peptide has reduced binding activity as compared to thewild-type SCP.
 3. The peptide of claim 1, wherein the peptide isexpressed from an isolated DNA sequence.
 4. The peptide of claim 1,wherein the SCP catalytic domain comprises a specificity crevice.
 5. Thepeptide of claim 4 wherein the SCP specificity crevice comprisescontiguous amino acid residues from residue 260 to residue 417, whereinthe position of the amino acid residues in the peptide corresponds tothe position of amino acids in wild-type SCP.
 6. The peptide of claim 1,wherein the SCP comprises one or more of amino acid residues 260, 261,262, 415, 416 or 417, wherein the position of the amino acid residues inthe peptide corresponds to the position of amino acids in wild-type SCP.7. The peptide of claim 1, wherein the SCP catalytic domain comprisescontiguous amino acid residues from residue 130 to residue 512, whereinthe position of the amino acid residues in the peptide corresponds tothe position of amino acids in wild-type SCP.
 8. The peptide of claim 1wherein the SCP catalytic domain comprises one or more of amino acidresidues 130, 193, 295 or 512, wherein the position of the ammo acidresidues in the peptide corresponds to the position of amino acids inwild-type SCP.
 9. The peptide of claim 1 wherein the peptide has asubstitution at one or more of amino acid residues 260, 261, 262, 415,416, 417, 130, 193, 295 or 512, wherein the position of the amino acidresidues in the peptide corresponds to the position of amino acids inwild-type SCP.
 10. The peptide of claim 9 wherein the substitution is aconserved substitution.
 11. The peptide of claim 9, wherein the peptideis SCPA49D130A, SCPA49H193A, SCPA49N295A, SCPA49S512A, SCPA1D130A,SCPA1H193A, SCPA1N295A, SCPA1S512A, SCPBD130A, SCPBH193A, SCPBN295A,SCPBS512A or ΔSCPA49.
 12. The peptide of claim 11, wherein the peptideis SCPA1S512A.
 13. The peptide of claim 1 wherein the peptide variesfrom wild-type SCP in that it does not contain a signal sequence. 14.The peptide of claim 1 wherein the peptide varies from wild-type SCP inthat it does not contain a cell wall insert.
 15. The peptide of claim 1wherein the SCP is from a group A Streptococcus, group B Streptococcus,group C Streptococcus or group G Streptococcus.
 16. The peptideaccording to claim 15, wherein the SCP is from Group A Streptococcus.17. The peptide of claim 8, wherein the peptide has a substitution atone or more of amino acid residues 130, 193, 295 or 512 with an alanine,valine, leucine, isoleucine, proline, phenylalanine, tryptophan ormethionine residue.